Disseminated Candidiasis caused by Candida albicans with amino acid substitutions in Fks1 at position Ser645 cannot be successfully treated with micafungin.

The clinical utility of the echinocandins is potentially compromised by the emergence of drug resistance. We investigated whether Candida albicans with amino acid substitutions at position Ser645 in Fks1 can be treated with either a conventional or an elevated dosage of micafungin. We studied Candida albicans (wild-type SC5314; MIC, 0.06 mg/liter) and four fks1 mutants (one FKS1/fks1 heterozygote mutant [MIC, 0.5 mg/liter] and three fks1/fks1 homozygous mutants [MICs for all, 2 mg/liter]) with a variety of amino acid substitutions at Ser645. The pharmacokinetic and pharmacodynamic relationships were characterized in a persistently neutropenic murine model of disseminated candidiasis. A mathematical model was fitted to all pharmacokinetic and pharmacodynamic data. This mathematical model was then used to "humanize" the murine pharmacokinetics, and the predicted antifungal effect was determined. The estimated maximal rate of growth and ultimate fungal densities in the kidney for each of the strains were similar. The administration of micafungin at 1 mg/kg of body weight to the wild type resulted in moderate antifungal activity, whereas the administration of 5 and 20 mg/kg resulted in rapid fungicidal activity. In contrast, the FKS1/fks heterozygote was killed only with 20 mg/kg, and the homozygous fks1 mutants failed to respond to any dosage. The bridging study revealed that human dosages of 100 and 400 mg/day were active only against the wild type, with no activity against either the heterozygote or the homozygote mutants. Ser645 Fks1 Candida albicans mutants cannot be treated with either conventional or elevated dosages of micafungin and should be deemed resistant.

Pathogenicity of Aspergillus fumigatus mutants assessed in Galleria mellonella matches that in mice.

Aspergillus fumigatus is a clinically important fungus with the ability to cause invasive aspergillosis with high mortality rates in immunocompromised patients and chronic pulmonary aspergillosis in immunocompetent individuals. Virulence of mutants has traditionally been assessed using mammalian hosts such as mice and rats and more recently the fruit fly, Drosophila melanogaster, demonstrated the potential to act as an in vivo host suitable for screening Aspergillus mutants. In this study using a larger thermotolerant invertebrate, Galleria mellonella, the virulence of individual gene deletants of Aspergillus fumigatus (cpcA, sidA, sidC, sidD, sidF and paba,) were compared to the parental and gene-replacement strains, if available. A range of infectious challenges consisting of from 3 × 10(3)-3 × 10(6) spores/larva was followed by observation of larval survival with mean survival time used as a surrogate of microbial pathogenicity. Mutants cpcA, sidA, sidF and paba were avirulent and sidC and sidD showed attenuated virulence. Virulence assessment in G. mellonella correlated closely with the historic data generated using mice and Drosophila. Pre-screening Aspergillus mutants using G. mellonella could significantly reduce the number of mammals required to assess changes in virulence.

The next generation of liposome delivery systems: recent experience with tumor-targeted, sterically-stabilized immunoliposomes and active-loading gradients.

Three topics are discussed. Enhanced anti-tumor efficacy of targeted doxorubicin-containing sterically-stabilized liposomes using an anti-beta1 integrin Fab' ligand. Use of tumor targeting with an internalizing ligand to improve the efficacy of a non-leaky cisplatin-containing sterically-stabilized liposome formulation. Formulation variables (remote-loading with dextran ammonium sulfate, rigid lipid bilayer) used to optimize in vivo performance of a liposomal camptothecin analog.

Solute-induced shift of phase transition temperature in Di-saturated PC liposomes: adoption of ripple phase creates osmotic stress.

We have examined the calorimetric behavior of large liposomes consisting of symmetric saturated chain phosphatidylcholines. Most notably, for systems made in solutions containing solute (e.g., NaCl, glucose, etc.) there was an additional major endotherm just below the main phase transition temperature. The new endotherm was found to represent a population of lipid whose main phase transition was shifted to lower temperature due to an induced osmotic stress across the membrane. Absent for isoosmotic systems, the osmotic stress was created when the liposome internal volume decreased, a consequence of the Lbeta' (gel) to Pbeta' (rippled) phase transition. That is, rippling of the membrane caused vesicle volume to decrease (> or = 28%) and because the free flow of water outward was restricted by solute, an osmotic gradient was created where none had existed before. The distribution of enthalpy between the new shifted Tm and the expected Tm correlated with the percent of lipid in the outer bilayer and it was concluded that only the outer bilayer sensed the induced stress. Internalized liposome structures were shielded, thus explaining the persistence of the expected Tm in preparations made in solute. The shift in Tm (deltaTm) was discrete and linearly dependent upon lipid chain length for the PC series di-17:0 (deltaTm approximately 1.4 degrees C) through di-20:0 (deltaTm approximately 0.6 degrees C), suggesting a structural change (i.e., lipid packing/orientation) was involved. Although freeze-fracture electron microscopy of stressed and unstressed bilayers revealed no differences in ripple periodicity there were differences in surface features and in vesicle shape. The fact that this phenomenon has gone unnoticed for MLVs is probably due to the fact that these systems are known to exclude solute and thus exist under osmotic compression.

Canine model of abdominal aortic aneurysm treated by endovascular graft implantation.

The purpose of this study was to monitor the effects of endovascular graft implantation on a canine model of aortic aneurysm. Aneurysms were created in 10 dogs by fascial patch angioplasty of the infrarenal aorta. In five dogs, aneurysm creation was immediately followed by insertion of an endovascular graft. Central aortic and aneurysm sac pressures were then measured by needle puncture. The remaining five dogs were left untreated, as controls. Angiography was performed after aneurysm creation, after endovascular graft implantation, and at 1 month and 3 months. Following insertion of an endovascular graft, mean (s.d.) systolic pressure was lower in the aneurysm sac (82.9 (20.20) mmHg) than in the adjacent aorta (113.4 (25.9) mmHg; P < 0.002) in all the treatment group. The effects on diastolic pressure and mean pressure were less pronounced. Aneurysm size was increased in all controls (25.2 (9.55)%) and decreased in all of the treated group (22.5 (11.7)%; P < 0.001). In conclusion this model of aortic aneurysm has two important characteristics' it has multiple collateral branches, and it grows. Insertion of an endovascular graft was associated with a reduction in aneurysm sac pressure, reduced aneurysm growth, and fibrosis of the space between the aneurysm sac and the graft.

Curvature dependent induction of the interdigitated gel phase in DPPC vesicles.

Ethanol causes biphasic melting behavior in saturated lecithins (Rowe (1983) Biochemistry 22, 3299-3305), a consequence of the formation of the stable interdigitated phase (Simon, S.A. and McIntosh, T.J. (1984) Biochim. Biophys. Acta 773, 169-172). The membrane systems studied to date have been large vesicle systems in which the membrane surface can be assumed to be locally planar. An immediate question arises as to whether surfaces of higher curvature interdigitate. To address this question we have prepared DPPC vesicles of varying diameters which we employed to determine the limiting size at which interdigitation occurs using ethanol as the inducer. We find that with decreasing vesicle size the concentration of ethanol necessary for the onset of interdigitation increases. Small isolated vesicles, at inducing concentrations of ethanol, do not stably interdigitate but rupture and coalesce into a viscous gel comprised of interdigitated lipid sheets. As discussed elsewhere (Ahl et al. (1992) Biophys. J. 243a) these sheets can be used as precursors for producing liposomes of large size and high internal volumes useful in drug delivery or modeling applications.

Interdigitated bilayer packing motifs: Raman spectroscopic studies of the eutectic phase behavior of the 1-stearoyl-2-caprylphosphatidylcholine/dimyristoylphosphatidylcholine binary mixture.

The thermotropic properties and acyl chain packing characteristics of multilamellar dispersions of binary mixtures of 1-stearoyl-2-caprylphosphatidylcholine (C(18):C(10)PC), an asymmetric chain species, and dimyristoylphosphatidylcholine (C(14):C(14)PC), a symmetric chain lipid, were monitored by vibrational Raman spectroscopy. In order to examine each component of the binary mixture separately, the acyl chains of the symmetric chain species were perdeuterated. As shown by differential scanning calorimetry, the mismatch in the gel phase bilayer thickness between the two lipid components generates a lateral phase separation resulting in two distinct gel phases, G(I) and G(II), which coexist over much of the composition range. The Raman data demonstrate that the mixed interdigitated phase (three chains per headgroup), analogous to single component phase behavior, is retained when the C(18):C(10)PC component act as a host for the G(I) gel phase. In contrast, the C(18):C(10)PC molecules exhibit partial interdigitation (two chains per headgroup) when they are included as guests within the C(14):C(14)PC host matrix to form the G(II) gel phase. Compared to pure C(14):C(14)PC bilayers at equivalent reduced temperatures, the host G(II) gel phase C(14):C(14)PC molecules exhibit an increased acyl chain order, while for the host G(I) gel phase the C(14):C(14)PC lipid species show increased intrachain disorder.

Evaluation of the polyethylene glycol-indirect antiglobulin test for routine compatibility testing.

All specimens received in the blood bank over a 5-month period for crossmatch or group and screen requests were tested in parallel by a polyethylene glycol-indirect antiglobulin test (PEG-IAT) and a low-ionic-strength saline (LISS)-IAT. The sera of 41 of 1471 patients had reactions, with 50 antibodies being detected. Ten antibodies reacted only on the PEG-IAT and 14 only by the LISS-IAT; the remaining 26 antibodies were detected by both methods. Of the antibodies that reacted only by the LISS-IAT, one (anti-Jka) was considered clinically significant, whereas five of the antibodies that reacted only by the PEG-IAT (1 anti-c, 2-Fya, 1-Jkb, and 1-S) were considered significant. Two antibodies of questionable clinical significance were detected only by the PEG-IAT. In 97 percent of the sera tested, no reaction was detected by either method. The PEG-IAT is an acceptable technique for routine compatibility testing.

Polymorphic phase behavior of lysophosphatidylethanolamine dispersions. A thermodynamic and spectroscopic characterization.

We have investigated the phase behavior of aqueous dispersions of a series of synthetic lysophosphatidylethanolamines as a function of the acyl chain length. Lysophosphatidylethanolamines exhibit phase polymorphism encompassing a well-ordered crystalline phase which may arise either from a metastable interdigitated lamellar gel phase or a metastable micellar phase. The time course of interconversion between these various phases have been outlined by observing the low temperature incubation time dependence of the calorimetric thermograms. We have determined differences in structure of these phases by Raman spectroscopy and 31P nuclear magnetic resonance spectroscopy. It appears that a principal contribution to this polymorphic phase behavior lies in the nature of headgroup hydration and headgroup-headgroup interactions.

Scanning calorimetry reveals a new phase transition in L-alpha-dipalmitoylphosphatidylcholine.

We report a new phase transition in fully hydrated dispersions of dipalmitoylphosphatidylcholine (DPPC). This new transition, called the sub-subtransition, exhibits a transition enthalpy of 0.25 kcal/mol with a Tm at 6.8 degrees C. Unlike the subtransition, no extended low temperature incubation is required to observe the sub-subtransition. This new sub-subgel (SGII) phase may be a precursor to the subgel (SGI) phase, and this discovery is discussed in relation to the current knowledge regarding the polymorphic gel phases of both ester- and ether-linked lipids with identical acyl chains.